Strategies to edit paralogous genes with CRISPR/Cas9
- Authors: Nemudryi A.A1,2,3, Malankhanova T.B1,2,3,4, Malakhova A.A1,2,3, Medvedev S.P1,2,3, Zakian S.M1,2,3
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Affiliations:
- Federal Research Center Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences
- Institute of Chemical Biology and Fundamental Medicine, the Siberian Branch of the Russian Academy of Sciences
- State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation
- National Research University Novosibirsk State University
- Issue: Vol 11, No 2 (2016)
- Pages: 87-94
- Section: Articles
- URL: https://genescells.ru/2313-1829/article/view/120594
- DOI: https://doi.org/10.23868/gc120594
- ID: 120594
Cite item
Abstract
The purpose of this study is to develop the strategies of CRISPR/Cas9 application to improve fidelity and specificity of this platform. Here we use a model system, which includes target gene and a paralogue - potential aim for off-target double-strand break induction. The study was carried on using Brattleboro rats embryonic fibroblasts which are homozygous for a mutation in arginine-vasopressin gene (target). The potential off-target gene is oxytocin gene: its DNA sequence is almost identical to that of arginine-vasopressin gene. To prevent off-target effect we designed several strategies, which were further used on Brattleboro rats embryonic fibroblasts. Here we show, that these strategies allowed us to generate double-strand breaks in arginine-vasopressin gene without any off-target effects in oxytocin gene. The endonuclease restriction assay shows that we have modified arginine-vasopressin gene while using both CRISPR/Cas9 and single-stranded oligonucleotides as a donor for homologous recombination. At last, if we consider Brattleboro rats as a model of monogenic disease the strategies designed could be translated in human therapeutic genome editing studies.
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About the authors
A. A Nemudryi
Federal Research Center Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences; Institute of Chemical Biology and Fundamental Medicine, the Siberian Branch of the Russian Academy of Sciences; State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation
T. B Malankhanova
Federal Research Center Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences; Institute of Chemical Biology and Fundamental Medicine, the Siberian Branch of the Russian Academy of Sciences; State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation; National Research University Novosibirsk State University
A. A Malakhova
Federal Research Center Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences; Institute of Chemical Biology and Fundamental Medicine, the Siberian Branch of the Russian Academy of Sciences; State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation
S. P Medvedev
Federal Research Center Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences; Institute of Chemical Biology and Fundamental Medicine, the Siberian Branch of the Russian Academy of Sciences; State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation
S. M Zakian
Federal Research Center Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences; Institute of Chemical Biology and Fundamental Medicine, the Siberian Branch of the Russian Academy of Sciences; State Research Institute of Circulation Pathology, Ministry of Healthcare of the Russian Federation
Email: zakian@bionet.nsc.ru
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